User:M Jaffe/Notebook/Maxis Protease Lab 471/2015/09/29: Difference between revisions

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==Procedure==
==Procedure==


#We retrieved one of our Trypsin eppendorf tube from our freezer box. We added 1 mL of 50mM Tris buffer 10 mM CaCl2at pH 8 to the tube and recorded the concentration which was 46.35 µM.
In order to do the Bradford Assay of Protease Degradation, we followed Dr. Hartings' [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2015/09/29|protocol]], and did the following specific steps:
#We weighed out 7 eppendorf tubes of dried gold fibers.
*Protease Sample Prep.
#We added 978.4 µL 50mM Tris buffer 10 mM CaCl2 at pH 8 to each of the 7 dried gold fiber tubes.  
#Used eppendorf tube no. 1 that weighed 1.0269g, and contained 0.00108g.
#We scrapped the gold fibers that were stuck to the sides of the eppendorf tube. We used the vortex on each tube for approximately 1 minute.
#Added 1mL of phosphate/CaCl2 buffer.
#We added 21.57 µL of Trypsin solution to each gold fiber and buffer solutions.
#Final concentration: 46.35µM
#We placed the 7 tubes of gold trypsin and buffer into a 37C water bath.
*Sample Prep.  
#We started the timer.
#Used 7 eppendorf tubes, each containing gold fibers.
#While the samples were heated we prepared Bradford assay in plastic cuvettes. We did a 1 to 4 dilution of Bradford to Tris buffer.
#To each tube add:
#Then we added the following to each cuvette.    
##0.9784mL of buffer
##600 uL of diluted Bradford
##0.02157mL of trypsin (add this at the time of putting the tubes in the hot water bath).
##1650 uL of buffer
*Blank Prep.  
#After each time interval, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hr, 1.5 hr, 2 hr., we removed the Trypsin samples from the water bath, centrifuged the sample for 1 min to pull any fibers to the bottom of the tube and then added 750µL to the already prepared cuvette.  
#In on eppendorf tube add:
#Then we ran the sample on the UV/Vis spectrophotometer, recorded and saved the data.
##0.9784mL of buffer
#We did steps 10-11 for all 7 samples.
##0.02157mL of trypsin
*Bradford Dilution
#Dilute 1mL of Bradford in 3mL of buffer such that the resulting solution is 1:4.
 
Calculations:
    V1 = [(1µM)(1mL)]/46.35µM = 0.02157mL, amount of trypsin solution needed
    Volume of buffer: 1mL - 0.02157mL = 0.9784mL
 
==Data==
 




Revision as of 17:47, 5 October 2015

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Objectives

Conduct Bradford analysis on protease (trypsin) degraded samples. Instructions can be found here.


Procedure

In order to do the Bradford Assay of Protease Degradation, we followed Dr. Hartings' protocol, and did the following specific steps:

  • Protease Sample Prep.
  1. Used eppendorf tube no. 1 that weighed 1.0269g, and contained 0.00108g.
  2. Added 1mL of phosphate/CaCl2 buffer.
  3. Final concentration: 46.35µM
  • Sample Prep.
  1. Used 7 eppendorf tubes, each containing gold fibers.
  2. To each tube add:
    1. 0.9784mL of buffer
    2. 0.02157mL of trypsin (add this at the time of putting the tubes in the hot water bath).
  • Blank Prep.
  1. In on eppendorf tube add:
    1. 0.9784mL of buffer
    2. 0.02157mL of trypsin
  • Bradford Dilution
  1. Dilute 1mL of Bradford in 3mL of buffer such that the resulting solution is 1:4.

Calculations: 

   V1 = [(1µM)(1mL)]/46.35µM = 0.02157mL, amount of trypsin solution needed
   Volume of buffer: 1mL - 0.02157mL = 0.9784mL

Data


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